Arsenic is a contaminant of drinking water that affects millions of people throughout the world. Exposure during embryonic development is associated with low birth weight, altered locomotor activity, and reduced neuronal development, likely because of reductions in the number or localization of skeletal myocytes and neurons. Indeed, our data indicate that arsenite exposure to stem cell-derived embryoid bodies reduces the expression of MyoD, Myf5, myogenin, and NeuroD, neurogenin-1, and neurogenin-2, all transcription factors needed to convert stem cells into skeletal myocytes and sensory neurons, respectively. Both of these cell types share a common lineage in the neural plate border specifier cells, which express transcription factors involved in myogenesis, such as Pax3, Pax7, Dlx5, Msx1, and Msx2, and are responsible for secreting factors that lead to the formation of the neural crest specifier cells, which produce sensory neurons. In embryonic bodies exposed to arsenite, Msx2 expression is reduced and Pax3 spatial localization within the embryoid body is altered. These data suggest that the number and spatial localization of neural plate border specifier cells may play a critical role in the developmental toxicity of arsenic. The goal of this application is to determine whether arsenic alters the number and/or localization of neural plate border specifier cells and to determine the specific inductive or repressive signaling pathway targeted by arsenic during the formation of the neural plate, neural crest, and the mesoderm. In the first aim, we will examine changes in the localization and number of the neural plate border specifier cells following exposure to arsenic or its methylated metabolites. These results will enable us to both determine if the specifier cells are altered, and also determine whether neural crest cell production is inhibited. The goal of the second aim is to determine the inductive and repressive signaling pathways that are targeted by arsenic. Specifically, we will use Wnt1, Wnt3, Wnt3a, Wnt5a, noggin, Bmp2, and Bmp4 to differentiate the embryonic stem cells and investigate whether arsenic abrogates sensory neuron and skeletal myotube formation. Our long-term objective is to understand why arsenic-exposed populations are at increased risk for defects in skeletal muscle and neuronal development, and how this leads to functional changes such as low birth weight and altered neuronal function. We can then use this generated data to investigate the specific mechanisms by which arsenic alters the development of neural crest cells and paraxial mesoderm.